Cell division rates decrease with age, providing a potential explanation for the age-dependent deceleration in cancer incidence Cristian Tomasettia,b,c,1, Justin Polingd, Nicholas J. Robertsb,e, Nyall R. London Jr.f, Meredith E. Pittmang, Michael C. Haffnerb,h, Anthony Rizzoh, Alex Barash, Baktiar Karimi, Antonio Kimb, Christopher M. Heaphyb,h, Alan K. Meekerb,h, Ralph H. Hrubanb,e,h,j,k, Christine A. Iacobuzio-Donahuel, and Bert Vogelsteinb,j,k,1 aDivision of Biostatistics and Bioinformatics, Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD 21205; bSidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, MD 21205; cDepartment of Biostatistics, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205; dPathology, Williamson Medical Center, Brentwood, TN 37207; eDepartment of Pathology, The Sol Goldman Pancreatic Cancer Research Center, The Johns Hopkins University, Baltimore, MD 21231; fDepartment of Otolaryngology, Johns Hopkins University School of Medicine, Baltimore, MD 21205; gDepartment of Pathology and Laboratory Medicine, Weill Cornell Medicine, New York, NY 10065; hDepartment of Pathology, The Johns Hopkins University, Baltimore, MD 21231; iPathology & Histotechnology Laboratory, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21702; jLudwig Center, Johns Hopkins University School of Medicine, Baltimore, MD 21205; kHoward Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21205; and lDepartment of Pathology, Rubenstein Center for Pancreatic Cancer Research, Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065 Contributed by Bert Vogelstein, July 15, 2019 (sent for review April 4, 2019; reviewed by Ronny Drapkin and Michel Wassef) A new evaluation of previously published data suggested to us that DNA repair processes improve in aged individuals, and no that the accumulation of mutations might slow, rather than evidence for such beneficial changes in repair has emerged. We increase, as individuals age. To explain this unexpected finding, considered it more likely that cell division rates decrease with we hypothesized that normal stem cell division rates might de- age. There is little known about division of stem cells in human crease as we age. To test this hypothesis, we evaluated cell division self-renewing tissues over time except that the number (rather rates in the epithelium of human colonic, duodenal, esophageal, than division rate) of hematopoietic stem cells and epidermal and posterior ethmoid sinonasal tissues. In all 4 tissues, there was stem cells appear not to decrease with age (17, 18). a significant decrease in cell division rates with age. In contrast, cell division rates did not decrease in the colon of aged mice, and Results only small decreases were observed in their small intestine or Cell Division Rates Decelerate in Human Tissues. To test the hy- esophagus. These results have important implications for un- derstanding the relationship between normal stem cells, aging, pothesis that cell division rates may actually decrease with age, and cancer. Moreover, they provide a plausible explanation for the histologically normal colon samples were obtained from 13 in- enigmatic age-dependent deceleration in cancer incidence in very dividuals in each of 2 age cohorts: 20 to 29 y of age and 80 to 89 y old humans but not in mice. of age. We used the Ki67 antibody, a well-established prolifer- ation marker, to label sections from these tissues (Materials and cell division | aging | cancer | mutation rate Methods). Ki67 labels nuclei during all active phases of the omatic mutation rates have been measured in several human Significance Scell types with a variety of techniques (1–5). For both nuclear and mitochondrial genomes, somatic mutations appear to in- We have discovered a species-specific feature of the aging crease in a linear fashion throughout life. On the surface, this process: significantly slowed division rates in self-renewing accumulation is consistent with the increase in tumor incidence tissues of humans. In contrast, we found only small to no de- with age that forms one of the cornerstones of our understanding crease in division rates in self-renewing tissues of mice, for of cancer (6, 7). This relationship between age and cancer in- whom cancer incidence does not decelerate with age. This cidence has important practical and conceptual ramifications, discovery has important implications for the process of aging given that the proportion of very old individuals in the pop- itself, particularly given that there is so little known about this ulation is expected to increase. As noted by many investigators, ubiquitous process. It additionally provides a potential expla- however, there is a blemish in this otherwise satisfying picture: In nation, supported by experimental data, for the enigmatic very old individuals, cancer incidence rates often decelerate (8– decrease of cancer in the oldest (and most rapidly growing) 11). Although hypotheses for this deceleration have been pro- segment of Western populations. posed, understanding of the deceleration of cancer incidence with age remains incomplete (8, 10–13). This conundrum led us Author contributions: C.T. conceived the study; C.T., C.A.I.-D., and B.V. designed research; to question whether the expected linear accumulation of mu- C.T., J.P., N.J.R., N.R.L., M.E.P., M.C.H., A.R., A.B., B.K., A.K., C.M.H., A.K.M., R.H.H., C.A.I.-D., and B.V. performed research; C.T., C.M.H., and A.K.M. contributed new re- tations with age continues throughout life, even in old indi- agents/analytic tools; C.T. and B.V. analyzed data; and C.T., J.P., N.J.R., N.R.L., M.E.P., viduals. While evaluating the literature on this topic (4, 14, 15), M.C.H., A.B., C.M.H., A.K.M., R.H.H., C.A.I.-D., and B.V. wrote the paper. we noted that it was generally assumed, rather than demon- Reviewers: R.D., University of Pennsylvania; and M.W., Institut Curie, Paris Sciences et strated, that the mutations continued to accumulate in a linear Lettres Research University. fashion over time. When we removed the assumption of line- The authors declare no conflict of interest. arity from such regression analyses, a different picture often Published under the PNAS license. seemed to emerge: a trend toward slowing in the rate at which Data deposition: All data are available on GitHub (https://github.com/cristomasetti/). SI Appendix – somatic mutations accumulate with age ( ,Figs.S1 1To whom correspondence may be addressed. Email: [email protected] or ctomasetti@ S3). This observation is supported by the analyses provided by jhu.edu. Podolskiy et al. (16) This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. How could this occur? It is unlikely that the DNA polymerases 1073/pnas.1905722116/-/DCSupplemental. responsible for replicating DNA acquire increased fidelity or First Published September 23, 2019. 20482–20488 | PNAS | October 8, 2019 | vol. 116 | no. 41 www.pnas.org/cgi/doi/10.1073/pnas.1905722116 Downloaded by guest on October 2, 2021 −3 cell cycle (G1,S,G2, and mitosis), but is absent from resting a 39% (95% CI = 13 to 65%; P < 4 · 10 ) reduction, re- cells (G0). spectively. These results, highly significant even when controlling We identified 151 labeled cells (15% of the 992 analyzed cells) for multiple testing, document that colonic crypt epithelial cell in the older cohort at positions 1 to 8 from the crypt bases—the proliferation is reduced in older individuals. region where the stem cells are thought to reside, whereas in the We also performed a fourth experiment where the colonic younger cohort, 219 labeled cells were found (26% of the 848 tissues were double-stained for Ki67 as well as Lgr5, a marker analyzed cells) (Fig. 1). The difference in Ki67 labeling was associated with colonic stem cells. The double-positive cells were − highly significant (P < 3 ·10 5), with the proliferation rate in the too few to obtain a statistically significant result, but a trend was older group reduced by 41% of that in the younger group (95% found for the younger colons having higher proliferation than confidence interval [CI] = 23 to 59%) (experiment 1A, Fig. 2). the older cohort, with 16 double-stained cells (out of 14,400 total Moreover, a similar pattern was observed throughout the cells counted [i.e., 0.001%]) in the old cohort vs. 56 double- replicating portion of the colonic epithelium (Dataset S1, re- stained cells (out of 18,416 total cells counted [i.e., 0.003%]) in ferring to experiment 1A). the young cohort (Materials and Methods and SI Appendix, A reanalysis performed by a second pathologist, using an au- Fig. S5). tomated imaging method for the counting of labeling in the same Finally, in colon, we performed a fifth experiment on 46 samples along the full crypt, confirmed these results: a highly paraffin-embedded sections of histologically normal colon (20 − significant 44% reduction (P < 5 · 10 7) in the older group samples in the younger cohort and 26 in the older cohort), where (95% CI = 28 to 60%) (experiment 1B, Fig. 2 and Materials the colonic tissues were labeled using an antibody raised against and Methods). the mitosis-specific phosphorylation of histone H3 protein To further validate these findings, experiments were per- (pHH3); this protein identifies dividing cells within the late G2 formed on 2 independent sets of histologically normal human phase to M phase of the cell cycle (19), and represents an al- colon tissues using the same labeling technique (experiments 2 ternative marker of cell proliferation. By analyzing the first 8 cell and 3, Fig. 2). In experiment 3, an antibody that recognizes the positions from the base of the crypts on each side, we identified lamin A/C protein was utilized to better visualize the outline of 14 labeled cells (1.02% of 1,376 cells analyzed) in the younger each nucleus, particularly the regions that have high cellularity cohort, and we identified 10 labeled cells (0.53% of the 1,872 (Materials and Methods and SI Appendix, Fig.